Our long term goal is to identify regions and specific amino acids within the steroidogenic P450s which play key roles in the unique substrate specificities of their hydroxylation reactions. In the adrenal cortex of most species two mitochondrial P450s (P450scc and P45011Beta) and two microsomal P450s (P45017alpha and P450C21) are involved in the biosynthesis from cholesterol of glucocorticoids (cortisol), mineralocorticoids (aldosterone) and the C19 androgen precursors of the sex hormones. Like most forms of P450 involved in endogenous substrate metabolism, these steroid hydroxylases demonstrate a high degree of substrate specificity both with respect to the steroids which they bind and the stereospecificity of their hydroxylation reactions. We will utilize mutagenesis and expression in bacteria to examine the requirements for both properties of these enzymes. We have developed procedures for relatively high level expression of functional forms of P45017alpha and P450scc in E. coli. Accordingly we propose specific mutagenic experiments to investigate these two steroid hydroxylases in this facile expression system which readily permits not only evaluation of enzymatic properties but also spectral characteristics (substrate-binding and heme-binding) of the mutant enzymes. The ability to easily examine the spectral properties of these enzymes in bacteria provides us the opportunity to estimate the effect of mutagenesis on the structural properties of steroid hydroxylase, as well as on their enzymatic properties. We will also develop E. coli systems expressing P45011beta and P450C21 to be used in similar mutagenesis experiments. In addition we will use the bacterial expression system to evaluate structure/function relationships of mutant forms of human P45017alpha from individuals having 17alpha-hydroxylase deficiency. We will also purify and characterize the cytosolic bacterial reductase which supports P45017alpha activities in intact bacteria. Finally we will develop procedures for purification of P45017alpha and P450scc from bacterial membranes to be used for detailed biophysical studies and crystallization. These studies will provide detailed information on the structure/function relationships of the adrenocortical steroid hydroxylases in particular and P450s in general and surely will broaden our understanding of this superfamily of gene products.